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In Vitropollen Germination of Orchids Traditionally Used 148 European Journal of Environmental Sciences IN VITRO POLLEN GERMINATION OF ORCHIDS TRADITIONALLY USED TO PRODUCE SALEP YASEMIN KEMEÇ1,*, KAAN HÜRKAN1, and CÜNEYT AKI2 1 Çanakkale Onsekiz Mart University, Institute of Natural and Applied Sciences, Department of Biology, 17100 Çanakkale, Turkey 2 Çanakkale Onsekiz Mart University, Faculty of Science and Arts, Department of Biology, 17100 Çanakkale, Turkey * Corresponding author: [email protected] ABSTRACT In Turkey the tubers of about 120 orchid species are widely collected for manufacturing the traditional drink salep. In this study, we focused on the in vitro germination of the pollen of the salep orchid species Ophrys mammosa, Orchis provincialis, Anacamptis morio subsp. morio, Orchis simia and Neotinea tridentata and discussed the potential effects this might have on the conservation of these orchids by reducing the need to collect them in the field. Pollen was sown on different media; Knudson, Orchimax and the medium described by Malmgren, and then incubated at 24 ± 1 °C in darkness for 24 h. Germinated pollen was stained with Brilliant Blue and examined under a stereoscopic microscope. Results of Tukey and Dunnett T3 statistical tests indicated that in terms of percentage germination, the best germination was observed on O. mammosa by 55% and Orchimax was the most successful medium by 50.5%. For pollinaria germination, the best rate was observed on O. mammosa by 69%. The medium Malmgren was the best germinative by 61.3%. It is clearly seen that difference in germination rates among studied species are achieved using different media. The development of such a method of studied species in this research points to the fact that this is possible and should serve as encouragement for others to devise procedures for other species. These kinds of researches on propagation of orchids would be useful to reintroducing some of the rarer, endangered and endemic species in Turkey such previously succeed for Orchis militaris and Liparis loeselii in Great Britain. Keywords: pollinium, pollinarium, orchid, in vitro germination, salep, conservation Introduction Around 120 orchid species, including the genera; Ace- ras, Anacamptis, Balia, Dactylorhiza, Himantoglossum, Orchids are cosmopolitan and occur in almost every Neotinea, Ophrys, Orchis and Serapias, are used to make habitat except in the Polar Regions (Edwards 2007). With salep in different places in Anatolia. Therefore, orchids more than 1000 genera and at least 25,000 species, the belonging to these genera are widely and intensively family Orchidaceae is the largest and the most diverse collected from nature. In this study, we focused on the family of flowering plants (Harrap and Harrap 2009). orchids Ophrys mammosa (Desf.), Orchis provincialis While nearly 70% of orchids live on other plants as ep- (Balb.), Anacamptis morio (L.) R. M. Bateman, Pridgeon iphytes, they can also grow in soil (25%), and live on and M. W. Chase subsp. morio, Orchis simia (Lam.) and rocks and decaying plants (5%) (Arditti 1979; Renz and Neotinea tridentata (Scop.) R. M. Bateman, Pridgeon Taubenheim 1984). In Turkey there are 170 terrestrial or- and M. W. Chase, which have been collected since an- chids belonging to 24 genera (Kreutz 2009). cient times in Anatolia and used to make salep (Özhatay In addition to their aesthetical and medicinal impor- et al. 1997; Baytop 1999). tance, orchids are ecological indicators (Joshi et al. 2009). Although orchids are protected worldwide from over- Moreover, orchids are used to manufacture a nutritious exploitation by the Convention on International Trade drink, called salep, which has been very popular for cen- in Endangered Species of Wild Fauna and Flora (CITES) turies in Anatolian and Arabian cultures and used as an (UNEP-WCMC 2013), they are still threatened by the additive in the production of “Maras” ice cream in Tur- illegal trade in orchids and the collecting of them from key (Sezik 1984; Baytop 1999; Kreutz 2009). nature to produce salep and “Maras” ice cream (Sezik et al. 2007; Kreutz 2009). It is estimated that 20–30 million orchids weighing 20 tons are collected from nature annu- Table 1 Tuber weights and tuber counts of orchids per 1000 kg (Sezik ally for producing salep (See Table 1). 1984). Because of the threat from logging, mining, urban- Commercial Average Number of tubers isation as a consequence of the increase in the human name tuber weight (g) per kg population, agricultural activities and the collecting of Muğla Salebi 0.23 4348 orchids from nature for the manufacture salep it is im- Kastamonu Salebi 0.50 2000 portant to develop new and advanced techniques for Silifke Salebi 0.35 2857 their sustainable usage (Kreutz 2009; Cribb 2011). This Antalya Salebi 0.21 4762 has resulted in improvements in the in vitro propagation of orchids in order to reduce the need to collect orchids Maraş Salebi 1.60 625 from nature and make it possible to conserve the most Van Salebi 1.00 1000 threatened species (Magrini et al. 2011). Thus, in this Kemeç, Y., Hürkan, K., Aki, C.: In vitro pollen germination of orchids traditionally used to produce salep European Journal of Environmental Sciences, Vol. 5, No. 2, pp. 148–152 http://dx.doi.org/10.14712/23361964.2015.88 In vitro pollen germination of orchids traditionally used to produce salep 149 Table 2 List of the species studied, locations, dates of collection and numbers of pollinaria collected. Collection location Collection of pollinaria Species (Çanakkale, Turkey) Date (2014) Number of pollinaria Anacamptis morio subsp. morio Kilitbahir April 15 210 Onsekiz Mart University Neotinea tridentata April 20 211 Terzioğlu Campus Ophrys mammosa Kilitbahir March 31 201 Orchis provincialis Denizgöründü village April 21 328 Orchis simia Denizgöründü village April 21 199 study, we developed a new method for the in vitro prop- M. W. Chase, growing wild in the centre of Çanakkale city agation using the pollen of orchids used in the manufac- and its surroundings (Turkey) (See Table 2 and Fig. 1). ture of salep. Plants were identified using “Flora of Turkey and the Although there are studies on the evolution (Brad- East Aegean Island” vol. 8 (Renz and Taubenheim 1984) shaw and Schemske 2003), ecology (Desrochers and and vol. 11 (Kreutz 2000), “Orkidelerimiz” (Sezik 1984) Rieseberg 1998), pollen ovule aging (Proctor 1998; Bel- and “Türkiye Orkideleri” (Kreutz 2009). lusci and Musacchio 2010), morphology and ultra-struc- ture of orchids (Feijo and Pais 1989; Pacini and Michael In vitro Germination of Pollen 2002) the germination pollen has not been previously Germination tests were started in March and April studied (Pritchard and Prendergast 1989; Aybeke 2002; 2014. The surfaces of pollinia were sterilized by placing Pacini and Michael 2002). them in a 0.5% solution of NaOC1 for 5 minutes after While many orchid genera have pollinia, which is an which they were rinsed three times in sterile distilled wa- adhesive mass of pollen, there are a few genera that pro- ter and between rinses they were broken into pieces in duce single pollen grains. Therefore, orchids differ great- a vortex mixer. ly in terms of whether they produce a mass of sticky pol- len or individual grains of pollen (Aybeke 2002). While the pollen of Pterostylis plumosa, Pterostylis concinna, Neottioids, Neuwiedia, Cypripedium acaule, Cypripedi- um calceolus, Apostasia wallachii consists of individual grains, that of Epipactis microphylla, Bletilla striata, Ne- ottia, Cleistes divaricata, Neottia nidus-avis, Epidendrum scutella, Epidendrum ibaguense, Loroglossum hircinum, Pleurothallis eumecocaulon and Calypso bulbosa con- sists of groups of four pollen grains (Pacini and Michael 2002). Using light-microscopy we observed that O. mam- mosa, O. provincialis, A. morio subsp. morio, O. simia and N. tridentata produce tetrad groups of pollens contained in pollinia. The aims of this study are to: (1) determine wheth- er it is possible to germinate the pollen of O. mammo- sa, O. provincialis, A. morio subsp. morio, O. simia and N. tridentata; (2) whether there differences in percentage germination between species and (3) the effects of KN, ORC and SV media on percentage germination. Material and Methods Collection of Study Materials Pollinia were collected from 5 species of orchids: Ophrys mammosa (Desf.), Orchis provincialis (Balb.), Fig. 1 Detailed images of flowers and pollinaria of N. tridentata and Anacamptis morio (L.) R. M. Bateman, Pridgeon and O. mammosa. Reproductive parts of Ophrys mammosa (A) and Neotinea M. W. Chase subsp. morio, Orchis simia (Lam.) and Neo- tridentata (C). Pollinarium of Ophrys mammosa (B) and Neotinea tinea tridentata (Scop.) R. M. Bateman, Pridgeon and tridentata (D); p: pollinium, c: caudicle, v: viscidium. European Journal of Environmental Sciences, Vol. 5, No. 2 150 Y. Kemeç, K. Hürkan, C. Aki Table 3 Species studied, their codes used in subsequent tables and numbers of sown pollinia and pollinaria on individual media. Number of sown pollinia Number of sown pollinaria Species Code KN ORC SV KN ORC SV Anacamptis morio subsp. morio A MOR 71 107 111 79 76 55 Neotinea tridentata N TRI 82 65 72 76 76 59 Ophrys mammosa O MAM 165 237 738 67 62 71 Orchis provincialis O PRO 119 84 121 113 123 92 Orchis simia O SIM 72 75 81 50 67 82 Two different commercial media (Orchimax Orchid were significant differences between N TRI, O MAM and Medium “ORC”, O0257 and Knudson C Orchid Medi- O SIM (Table 4). Overall germination rates of individ- um “KN”, K0215, Duchefa Biochemie BV, Haarlem, the ual media for pollinia are shown in Table 5. There were Netherlands) and one special medium (SV) (Malmgren no significant differences in germination rates of pollinia 2006) were used. KN and ORC media were supplement- among media (Table 5). Overall pollinia germination ed with 20 g/l sucrose, 1 g/l activated charcoal and 6 g/l rates for individual species are shown in Table 6.
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